MUTATED tRNA FOR CODON EXPANSION

Abstract

In some embodiments, the present disclosure relates to mutated tRNAs in which the first letter of the anticodon has been substituted to lysidine or agmatidine, and translation systems containing the mutated tRNAs. In a specific embodiment, the present disclosure provides mutated tRNAs capable of selectively translating codon NNA. In another embodiment, the present disclosure provides translation systems capable of translating two or three types of amino acids from a single codon box. In still another embodiment, the present disclosure provides novel methods for synthesizing lysidine diphosphate, agmatidine diphosphate, and derivatives thereof.

1 . A mutated tRNA produced by engineering a tRNA, wherein the engineering comprises a engineering such that, in its anticodon represented by N 1 N 2 N 3 , the first letter nucleoside N 1  after the engineering is any one of lysidine (k2C), a lysidine derivative, agmatidine (agm2C), and an agmatidine derivative, wherein N 2  and N 3  are arbitrary nucleosides for the second letter and the third letter of the anticodon, respectively,
 wherein the mutated tRNA comprises an anticodon complementary to a codon represented by M 1 M 2 A (wherein M 1  and M 2  represent nucleosides for the first and second letters of the codon respectively; each of M 1  and M 2  is selected from any of adenosine (A), guanosine (G), cytidine (C), and uridine (U); and the nucleoside of the third letter is adenosine), and   wherein M 1  and M 2  are selected from codons that constitute a codon box in which a codon with the third letter nucleoside being A and a codon with the third letter nucleoside being G both encode the same amino acid in the natural genetic code table.   
     
     
         2 .- 4 . (canceled) 
     
     
         5 . The mutated tRNA of  claim 1 , wherein the anticodon is represented by k2CN 2 N 3  or agm2CN 2 N 3  (wherein the nucleoside of the first letter of the anticodon is lysidine (k2C) or agmatidine (agm2C), and the nucleoside of the second letter (N 2 ) and the nucleoside of the third letter (N 3 ) are complementary to M 2  and M 1 , respectively). 
     
     
         6 . (canceled) 
     
     
         7 . The mutated tRNA of  claim 1 , wherein M 1  and M 2  are selected from codons that constitute a codon box in which a codon with the third letter nucleoside being U, a codon with the third letter nucleoside being C, a codon with the third letter nucleoside being A, and a codon with the third letter nucleoside being G all encode the same amino acid in the natural genetic code table. 
     
     
         8 . The mutated tRNA of  claim 1 , wherein M 1  and M 2  are selected from the group consisting of the following:
 (i) M1 is uridine (U) and M2 is cytidine (C);   (ii) M1 is cytidine (C) and M2 is uridine (U);   (iii) M1 is cytidine (C) and M2 is cytidine (C);   (iv) M1 is cytidine (C) and M2 is guanosine (G);   (v) M1 is guanosine (G) and M2 is uridine (U);   (vi) M1 is guanosine (G) and M2 is cytidine (C); and   (vii) M1 is guanosine (G) and M2 is guanosine (G).   
     
     
         9 . The mutated tRNA of  claim 1 , wherein an amino acid or an amino acid analog is attached to the 3′ end. 
     
     
         10 . A translation system comprising a plurality of different tRNAs, wherein the system comprises the mutated tRNA of  claim 1 . 
     
     
         11 . The translation system of  claim 10 , comprising (a) the mutated tRNA and (b) a tRNA comprising an anticodon complementary to a codon represented by M 1 M 2 G. 
     
     
         12 . The translation system of  claim 10 , further comprising (c) a tRNA comprising an anticodon complementary to a codon represented by M 1 M 2 U or M 1 M 2 C. 
     
     
         13 . The translation system of  claim 12 , wherein the amino acids or the amino acid analogs attached to the tRNAs of (a), (b), and (c) are different from one another. 
     
     
         14 . A method for producing a peptide, comprising translating a nucleic acid using the translation system of  claim 10 . 
     
     
         15 . A nucleic acid-peptide complex comprising a peptide and a nucleic acid encoding the peptide, wherein the nucleic acid encoding the peptide comprises the three codons of either (A) or (B) below:
 (A) M 1 M 2 U, M1M2A, and M1M2G;   (B) M1M2C, M1M2A, and M1M2G;   
       and wherein the amino acids corresponding to the three codons are all different on the peptide. 
     
     
         16 . The mutated tRNA of  claim 1 , wherein the number of nucleosides engineered is 20 or less. 
     
     
         17 . The mutated tRNA of  claim 1 , wherein the nucleic acid sequence of the engineered tRNA has sequence identity of 90% or more as compared to the nucleic acid sequence before the engineering. 
     
     
         18 . The mutated tRNA of  claim 1 , wherein the tRNA is one or more selected from the group consisting of tRNA Ala, tRNA Arg, tRNA Asn, tRNA Asp, tRNA Cys, tRNA Gln, tRNA Glu, tRNA Gly, tRNA His, tRNA Ile, tRNA Leu, tRNA Lys, tRNA Met, tRNA Phe, tRNA Pro, tRNA Ser, tRNA Thr, tRNA Trp, tRNA Tyr, and tRNA Val, tRNA fMet, tRNA Sec, tRNA Pyl, and tRNA AsnE2. 
     
     
         19 . The translation system of  claim 10 , wherein the mutated tRNA may be assigned to codons that constitute multiple codon boxes. 
     
     
         20 . The translation system of  claim 10 , which can translate more than 20 amino acids. 
     
     
         21 . The method for producing a peptide of  claim 14 , wherein the number of amino acids or amino acid analogs contained in the peptide is 9 or more and also 12 or less. 
     
     
         22 . The method for producing a peptide of  claim 14 , wherein the peptide contains an N-substituted amino acid(s). 
     
     
         23 . The method for producing a peptide of  claim 14 , wherein the peptide comprises a cyclic portion. 
     
     
         24 . The method for producing a peptide of  claim 23 , wherein the number of amino acids in the cyclic portion is 9 or more and also 11 or less.